Evaluation Of Different Extenders For The Cryopreservation Of Buffalo Bull (Bubalus Bubalis) Semen

Material type: BookPublisher: 2011Subject(s): Department of Physiology | Phd. thesisDDC classification: 1538,T Dissertation note: Buffalo is playing an important role in our country's economy by producing milk, meat and draught power. Genetic potential of low producing animals can be improved by using artificial insemination technology. Unfortunately, less number of elite bulls are available and low fertility rate of buffalo by using cryopreserved semen has been obtained. Semen is exposed to osmotic and oxidative stresses during processing, cryopreservation and thawing before insemination. Fertilizing ability is lost due to spermatozoa damage and it ultimately results in poor conception rates in buffalo. In order to protect spermatozoa from these stresses and improve fertility in buffalo, five osmotic pressure based concentrations of three extenders i.e. Citrate egg yolk extender (CEYE), Tris egg yolk extender (TEYE), and Lactose egg yolk extender (LEYE) were prepared by varying the quantity of the solutes to obtain an osmotic pressure of 255, 265, 275, 285 and 295 mOsm/kg. Osmotic pressure was measured by an osmometer.
In the first experiment, equal volume of semen obtained from four Nili-Ravi buffalo bulls was pooled and used to study the effects of osmotic pressure on post thawed semen characteristics. For this purpose, three basic media: citrate fructose media, tris citric acid fructose media and lactose media were prepared and divided each media in to five equal parts to maintain osmotic pressures of 255, 265, 275, 285 and 295mOsm/kg. These basis media were stored in a biomedical freezer, which were later used in preparing three semen extenders i.e. Citrate egg yolk extender (CEYE), Tris egg yolk extender (TEYE), and Lactose egg yolk extender (LEYE). During each collection, fifteen extenders (each of three extenders having five osmotic pressures i.e. 255, 265, 275, 285 and 295mOsm/kg) were used to extend the semen. After freezing, semen characteristics like sperm motility rate, viability rate, acrosomal integrity rate, plasma membrane integrity (PMI) rate, MTT reduction rate, sperm DNA integrity rate and lipid peroxidation were noted. Post thaw sperm motility rate in (%) CEYE was significant (P<0.05) at 295mOsm/kg compared to 255, 265 and 275mOsm/kg. However, sperm motility rate of different osmotic pressures of TEYE and LEYE was non-significant (P>0.05). Sperm viability rate (%) was non-significant (P>0.05) in all three extenders. Sperm acrosomal integrity rate was non-significant in CEYE and LEYE. However, it was significant (P<0.05) at 265, 275 and 295mOsm/kg in TEYE. Sperm PMI rate, MTT reduction rate, sperm DNA integrity rate and lipid peroxidation were non-significant (P>0.05) in CEYE, TEYE and LEYE.
On the basis of the individual and overall comparison of different semen characteristics of three extenders and their osmotic pressures, LEYE with 295mOs.kg was considered to be continued in the next experiment to upgrade the extender by adding taurine (TA) at 0.0, 30, 50 and 70 mM and trehalose (TR) at 0.0, 20, 40, 60 mM concentration. Semen collection, processing, freezing etc were done as per experiment-1 and same post thaw tests were carried out. Post thaw sperm motility rate was significantly (P<0.05) higher at TA-0.0 and TA-20mM and all concentration of TR. Sperm viability rate, acrosomal integrity rate, PMI rate, MTT reduction rate and lipid peroxidation at different concentrations of TA and TR were recorded non-significant (P>0.05). However, sperm DNA integrity rate was significant (P<0.05) higher at TA-0.0 and TR-0.0mM.
On the basis of comparison of different semen characteristics under various concentrations of taurine or trehalose, supplemented in semen extenders. Concentration of TR-70mM was considered to be continued in the next experiment to test fertility of the optimized extender.
Semen straws of LEYE supplemented with TR-70mM were used to inseminate the 50 buffaloes in heat (Supplemented group), while, traditionally used tris based buffalo bull semen extender was used (control group) to compared pregnancy rate (PR) of this experiment. Pregnancy rate in control and supplemented group was 38 and 54% respectively, which was statistically non-significant (P>0.05).

Buffalo is playing an important role in our country's economy by producing milk, meat and draught power. Genetic potential of low producing animals can be improved by using artificial insemination technology. Unfortunately, less number of elite bulls are available and low fertility rate of buffalo by using cryopreserved semen has been obtained. Semen is exposed to osmotic and oxidative stresses during processing, cryopreservation and thawing before insemination. Fertilizing ability is lost due to spermatozoa damage and it ultimately results in poor conception rates in buffalo. In order to protect spermatozoa from these stresses and improve fertility in buffalo, five osmotic pressure based concentrations of three extenders i.e. Citrate egg yolk extender (CEYE), Tris egg yolk extender (TEYE), and Lactose egg yolk extender (LEYE) were prepared by varying the quantity of the solutes to obtain an osmotic pressure of 255, 265, 275, 285 and 295 mOsm/kg. Osmotic pressure was measured by an osmometer.
In the first experiment, equal volume of semen obtained from four Nili-Ravi buffalo bulls was pooled and used to study the effects of osmotic pressure on post thawed semen characteristics. For this purpose, three basic media: citrate fructose media, tris citric acid fructose media and lactose media were prepared and divided each media in to five equal parts to maintain osmotic pressures of 255, 265, 275, 285 and 295mOsm/kg. These basis media were stored in a biomedical freezer, which were later used in preparing three semen extenders i.e. Citrate egg yolk extender (CEYE), Tris egg yolk extender (TEYE), and Lactose egg yolk extender (LEYE). During each collection, fifteen extenders (each of three extenders having five osmotic pressures i.e. 255, 265, 275, 285 and 295mOsm/kg) were used to extend the semen. After freezing, semen characteristics like sperm motility rate, viability rate, acrosomal integrity rate, plasma membrane integrity (PMI) rate, MTT reduction rate, sperm DNA integrity rate and lipid peroxidation were noted. Post thaw sperm motility rate in (%) CEYE was significant (P<0.05) at 295mOsm/kg compared to 255, 265 and 275mOsm/kg. However, sperm motility rate of different osmotic pressures of TEYE and LEYE was non-significant (P>0.05). Sperm viability rate (%) was non-significant (P>0.05) in all three extenders. Sperm acrosomal integrity rate was non-significant in CEYE and LEYE. However, it was significant (P<0.05) at 265, 275 and 295mOsm/kg in TEYE. Sperm PMI rate, MTT reduction rate, sperm DNA integrity rate and lipid peroxidation were non-significant (P>0.05) in CEYE, TEYE and LEYE.
On the basis of the individual and overall comparison of different semen characteristics of three extenders and their osmotic pressures, LEYE with 295mOs.kg was considered to be continued in the next experiment to upgrade the extender by adding taurine (TA) at 0.0, 30, 50 and 70 mM and trehalose (TR) at 0.0, 20, 40, 60 mM concentration. Semen collection, processing, freezing etc were done as per experiment-1 and same post thaw tests were carried out. Post thaw sperm motility rate was significantly (P<0.05) higher at TA-0.0 and TA-20mM and all concentration of TR. Sperm viability rate, acrosomal integrity rate, PMI rate, MTT reduction rate and lipid peroxidation at different concentrations of TA and TR were recorded non-significant (P>0.05). However, sperm DNA integrity rate was significant (P<0.05) higher at TA-0.0 and TR-0.0mM.
On the basis of comparison of different semen characteristics under various concentrations of taurine or trehalose, supplemented in semen extenders. Concentration of TR-70mM was considered to be continued in the next experiment to test fertility of the optimized extender.
Semen straws of LEYE supplemented with TR-70mM were used to inseminate the 50 buffaloes in heat (Supplemented group), while, traditionally used tris based buffalo bull semen extender was used (control group) to compared pregnancy rate (PR) of this experiment. Pregnancy rate in control and supplemented group was 38 and 54% respectively, which was statistically non-significant (P>0.05).

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